Method for making density gradients

ABSTRACT

A float is used for preparing a density gradient in a parallel-walled vessel. The float has an outer peripheral surface that has a diameter smaller than an inner diameter of an inner surface of the vessel. With the float placed inside the vessel a liquid is introduced onto the float such that the liquid flows around the float between the float and the inner wall of the vessel. The shape and configuration of the float slows the velocity of the liquid such that there is only laminar flow as the liquid contacts other liquid below the float. Elimination of turbulent flow prevents mixing of different liquid introduced into the same vessel thereby forming layers of fluid. Preferably, the vessel is a centrifuge tube. In one embodiment, the outer diameter of the float is large enough to cause capillary action between the float and the inner surface of the centrifuge tube to force liquid to remain between the float and the inner surface of the centrifuge tube.

This application is a divisional of U.S. patent application Ser. No.09/551,314, filed Apr. 18, 2000, now abandoned.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to an apparatus and method for making amultiple density layers or gradients of fluid in a vessel in a highlyreproducible manner using a float that floats on the surface of thefluid within the vessel.

B. Description of the Related Art

There are various fields where it is desirable to have density layers orgradients of fluid within a vessel for such purposes as the separationof matter, determining density, etc. Such density layers include, forexample, a solution retained in a vessel where the fluid is divided intoa plurality of layers, each layer having differing concentrations of asoluble material or solute. For example, a bottom or first layer offluid may have a concentration of a solute that is X moles per liter; asecond layer immediately above the first layer may have a concentrationof 0.8X moles per liter; a third layer above the second layer may have aconcentration of 0.6X moles per liter; and a fourth layer having aconcentration of 0.4X moles per liter.

Liquids having gradients of temperature, concentration, density andcolor have been previously prepared. Liquid density gradients have beenused for many years, for a wide variety of purposes, in a number ofdifferent industries. The inventor has numerous publications and patentsregarding certain aspects of gradient formation and use includingAnderson, N. G. Mechanical device for producing density gradients inliquids. Rev. Sci. Instr. 26: 891-892, 1955; Anderson, N. G., Bond, H.E., and Canning, R. E. Analytical techniques for cell fractions. I.Simplified gradient elution programming. Anal. Biochem. 3: 472-478,1962; Anderson, N. G., and Rutenberg, E. Analytical techniques for cellfractions. A simple gradient-forming apparatus. Anal. Biochem. 21:259-265, 1967; Candler, E. L., Nunley, C. E., and Anderson, N. G.Analytical techniques for cell fractions. VI. Multiplegradient-distributing rotor (B—XXI). Anal. Biochem. 21: 253-258, 1967.

A variety of other methods for making density gradients have beendeveloped, and Bock, R. M. and Ling, N.-S., Anal. Chem. 26, 1543, 1954,and Morris, C.J.O.R, and Morris, P., Separation Methods in Biochemistry,Pitman Publishing, 2nd ed. (1976) have reviewed many of these. Only oneof these methods allowed gradients to be made from multiple solutions,each having a different combination of reagents (Anderson, et al,“Analytical Techniques for Cell Fractions. I. Simplified GradientElution Programming”, Analytical Biochemistry 3: 472-478, 1962) Morerecent innovations include the use of pumps and pistons, which aredifferentially controlled by microprocessors, e.g., the Angeliquegradient maker (Large Scale Proteomics Corp. Rockville, Md.). Gradientsmay also be generated during high speed centrifugation by sedimenting agradient solute such as cesium chloride or an iodinated x-ray contrastmedium such as iodixanol. Gradients may be initially prepared as stepgradients and linearized by diffusion, by gentle mixing, or by freezingand thawing. A list of references covering existing methods follows.

Density gradients are used to make two basic types of separations. Thefirst separates particles on the basis of sedimentation rate (rate-zonalcentrifugation), in which case particles are separated on the basis ofthe size and density (and to a lesser extent shape) and particles willsediment farther if centrifuged for a longer period of time. The secondseparates particles on the basis of isopycnic banding density, in whichcase particles reach their equilibrium density level, and do notsediment farther with continued centrifugation.

Four types of gradients are in general use with either of these basicmethods. The first includes step gradients, made by layering a series ofsolutions of decreasing density (if the solutions are introduced oneabove the other), and of increasing density (if the solutions areintroduced sequentially to the bottom of the tube). The second typecomprises linear continuous gradients usually made by a mechanicalgradient maker. These are usually introduced slowly through small tubingto the bottom of the centrifuge tube. Linear gradients for either ratezonal or isopycnic zonal centrifugation are useful for resolving veryheterogeneous mixtures of particles.

The third type of gradient is non-linear, and may be designed toseparate particles having a very wide range of sizes or densities.Non-linear gradient may be designed to separate particles on the basisof both sedimentation rate and isopycnic banding density in the samegradient, in which case some particles reach their isopycnic level atsome point in the gradient, while others are still sedimenting.Generally such combined separations involve larger and denser particleswhich band near the bottom of the gradient, while other smaller, andusually lighter particles are still sedimenting in the upper portion ofthe gradient.

The fourth type of gradient is generated in a high centrifugal field bysedimentation of the major gradient solute, and is usually used forisopycnic banding.

Many reasons exist for desiring to control gradient shape. Gradientcapacity (i.e., the mass of particles which can exist in a zone withoutcausing a density inversion) is a function of gradient slope, and asteep gradient can support a greater mass of particles per unit gradientlength than can shallow gradients. The greatest particle massconcentration in a gradient separation usually occurs immediatelybeneath the sample zone shortly after centrifugation is started. Asdifferent particles separate in the length of the gradient, thepossibility of an overloaded zone diminishes. For this reason it isdesirable to have a short steep gradient section immediately under thesample zone, where the highest gradient capacity is required.

An additional reason for desiring to control gradient shape is that whena population of particles is present that differ little in sedimentationrate, these can best be separated by sedimentation through a longershallower section of the gradient. Such shallow sections are usuallynear the center of a gradient.

In the majority of density gradient separations, the gradients and theirchemical composition are designed to optimize the separation of one or afew particles types. This accounts for the very large number ofdifferent gradient recipes that have been published for subcellularfractionation. Those used for the isolation of mitochondria, forexample, are usually quite different from those used to isolate nuclei.For example, traces of divalent cations are required to control nuclearswelling, whereas such ions are generally deleterious to othersubcellular particles. Low concentrations of nonionic detergents removecytoplasmic contamination from nuclei, but are deleterious to theendoplasmic reticulum. Hence there has been no one procedure or gradientthat has been optimized for the systematic separation of the majority ofall subcellular particles. There is a need for reproducible means forincluding in gradients zones containing salts, detergents, enzymes andother reactive substances that would increase the number of differentsubcellular particles separated in one gradient.

Density gradient separations are important in proteomics research. Highresolution two-dimensional electrophoresis (2DE) is widely used toproduce global maps of the proteins in extracts prepared by solubilizingwhole cells or tissues. By careful control of the procedures employed,use of staining procedures which are quantitative, and computerizedimage analysis and data reduction, quantitative differences in theabundance of individual proteins of ±15% has been achieved (Anderson, N.Leigh, Nance, Sharron L., Tollaksen, Sandra L., Giere, Frederic A., andAnderson, Norman G., Quantitative reproducibility of measurements fromCoomassie Blue-stained two-dimensional gels: Analysis of mouse liverprotein patterns and a comparison of BALB/c and C57 strains.Electrophoresis 6: 592-599, 1985; Anderson, N. Leigh, Hofmann,Jean-Paul, Gemmell, Anne, and Taylor, John, Global approaches toquantitative analysis of gene-expression patterns observed by use oftwo-dimensional gel electrophoresis. Clin. Chem. 30: 2031-2036, 1984).There is a need for precision subcellular fractionation that will allowchanges in abundance of minor proteins to be accurately detected andmeasured in data which sums the abundance of all proteins found in allof the fractions of one sample.

This technology allows changes in gene expression, as reflected inprotein abundance, to be studied under a wide range of conditions, andhas led to the development of databases of protein abundance changes inresponse to a wide variety of drugs, toxic agents, disease states. Insuch studies large sets of data must be acquired and intercompared.Hence all stages in one pharmaceutical study, for example, must bestandardized for intercomparability.

2DE maps of whole cells or tissues typically contain a thousand or moreprotein spots in sufficient abundance to allow each protein to beanalyzed by mass spectrometry and identified and characterized. However,it is known that a very much larger number of proteins are actuallypresent in tissue samples analyzed than are actually observed. Thenumber present varies with cell or tissue type, and is believed to be upto ten or twenty times the number detected.

Different subcellular particles and the soluble fraction of the cell(the cytosol) contain many location-specific proteins which constituteonly trace fractions of the total cell protein mass. Hence the totalnumber of proteins resolved from one cell type or tissue could begreatly increased if the 2DE analysis were done on cell fractions ratherthan on whole cell or tissue extracts as has previously beendemonstrated (Anderson, N. L., Giere, F. A., et al, Affects of toxicagents at the protein level: Quantitative measurements of 213 mouseliver proteins following xenobiotic treatment. Fundamental and Appl.Tox. 8: 39-50, 1987). If a drug effect study is to be done on cellfractions, however, the fractionation procedures must be quantitative,in the sense that the same organelles, or even mixtures of organellesare used in all analyses to be intercompared. There exists, therefore,an emerging need for high resolution density gradient separations usingprecision gradients in proteomics research. Making precision gradientsreproducibly and in parallel has proven to be difficult, particularlywhen the gradients are shallow.

The protein composition of tissues such as liver varies diurnally, henceall the tissues from one group of animals are prepared at the same timeof day, and, to be comparable, must be fractionated in parallel, on thesame time schedule, and, if gradients are to be used, in identicalgradients. Further, gradient fraction recovery must also be done fromall gradients in parallel, under identical conditions. If the initialseparations are done partly or entirely on a sedimentation rate basis,and if the recovered fractions are to then each be isopycnically banded,as is done in two-dimensional or s-ρ fractionation, then thesesubsequent steps must also be carried out in parallel. This, in turn,requires that the gradients be made in parallel.

Precision gradients are difficult to make in practice, and it is furtherdifficult to confirm that a set of gradients are all identical withoutdestroying them for analysis. Existing swinging bucket rotors generallyallow six gradients to be centrifuged simultaneously. Larger numbers maybe centrifuged if the lower resolution of vertical or near vertical tuberotors is accepted. Therefore if existing density gradient formers areto be used, a set of six or more of them operating in parallel will berequired.

With any gradient maker, small amounts of turbulence or non-laminar flowtypically cause solutions of differing concentrations to at leastpartially mix, thereby reducing the effectiveness and usefulness of thedensity layers. There is therefore a need for a method for deceleratingfluids flowing into a tube, and for moving them slowly into position toform distinct bands.

One of many uses of density layers and gradients is in the fields ofcell separation, sub-cellular fractionation and analysis, and densitygradient methods are used in molecular biology and in polymer chemistry.Little attention has been paid to forming sets of precision-madegradients that are highly reproducible for cell separation. There istherefore a requirement for precision gradients adapted to cellseparation.

One high resolution system is disclosed in “Development of ZonalCentrifuges”, by N. G. Anderson, National Cancer Inst. Monograph 21,1966) and employs zonal centrifuge rotors. The rotors are of highcapacity, and process one sample at a time. However, the rotor volumesare too high for many applications. Angle head or vertical rotor tubesmay also be employed (Sheeler, P., Centrifugation in Biology andMedicine, Wiley Interscience, N.Y., 1981, 269pp) using either step orcontinuous gradients. However these do not provide the resolutionobtained with swinging bucket rotors.

There has been no reliable method for reproducibly locating andrecovering organelle zones purely on the basis of the physicalparameters of sedimentation rate and isopycnic banding density.Mathematical analyses, based on analysis not only of the biologicalparticles separated, but of the gradients themselves have been required.These have been tedious and idiosyncratic to the rotors and conditionsemployed. The basic problem in preparing density gradients in tubes isthat the liquid volume elements of either step (layers), or continuousgradients must be introduced into tubes very slowly or mixing willoccur. This problem is only partially overcome by introducing thegradient into a set of tubes in an angle-head rotor during rotation.

Methods for producing one or a few gradients in parallel have beendeveloped, but fraction recovery is generally done one at a time. Thegradients are rarely identical, and it is difficult to introduce thesample layer on top of the gradient without mixing. Hence there is nopublished data on the quantitative high-resolution protein analysis ofcell fractions of animals subject to various experimental treatments. Ifmultiple, parallel identical gradients are to be prepared using gradientengines (for instance, see “Mechanical device for producing densitygradients in liquids” by N. G. Anderson, Rev. Sci. Instruments 26:891-892, 1955) one must have one machine for each tube being filled.Centrifugal gradient distributing heads have been built (see “A MethodFor Rapid Fractionation of Particulate Systems by Gradient DifferentialCentrifugation” by J. F. Albright, and N. G. Anderson, Exptl. CellResearch 15: 271-281, 1958), however the gradients actually producedtend to be uneven, and a refrigerated centrifuge is required. There is,therefore, a continuing need for simple gradient makers that produceidentical gradients in parallel in sufficient number to satisfy currentrequirements. There is a further need for a simple, disposable andeasily sterilizable system for making reproducibly sharp step gradients.An additional need exists for a system or device that can produce verynarrow-step density gradients in which diffusion can rapidly andreproducibly even out the steps. A further need exists for a system ordevice which allows individual gradient steps to be rapidly pipettedinto centrifuge tubes, either manually or robotically, and in which theintroduced fluid does not disturb the underlying gradient. A stillfurther need exists for a gradient making device in which thecomposition of the successive layers, while forming a stable densityseries, differ in composition relative to salts, enzymes, detergents orother reactive materials.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a rapid, simple andreproducible method and apparatus for forming a multiplicity of liquiddensity gradients in vessels.

Another object of the present invention is to provide a rapid, simpleand reproducible method and apparatus for forming a multiplicity ofliquid density gradients in vessels for rate-zonal separations, forisopycnic banding separations, or a combination of the two.

Yet another object of the present invention is to provide an apparatusand method for reproducibly producing a plurality of liquid densitygradients in a plurality of corresponding vessels, each vessel having aspecific predetermined liquid density gradient.

An additional object of the present invention is to provide means formaking liquid density gradients in which aliquots of a liquid densityseries are rapidly pipetted into the centrifuge tubes without regard topotential stirring or mixing.

A further object of the invention is to decelerate the aliquots ejectedfrom pipettes or automatic pipetters, and to cause them to flow evenlyinto position without disturbing the underlying fluids.

A further object of the present invention is to provide means for makingthe linear or complex gradients by making them initially as stepgradients having very small density differences per step.

A further object of the present invention is to produce step gradientsin which the steps are so small that diffusion rapidly evens out thegradient.

A still further object of the present invention is to make the gradientmaking components disposable and easily sterilizable.

It is a further object of the present invention to make possibleconstruction of sets of identical gradients in a short period of time.

It is an additional object of the present invention to make possibleaddition of the sample layer on top of the gradient at any time afterthe gradient is formed.

In accordance with one aspect of the present invention, there is amethod for producing liquid density gradients in a vessel using a floatwithin the vessel includes the steps of:

inserting the float in the vessel;

introducing a first liquid into the vessel;

introducing a second liquid into the vessel such that the second liquidcontacts at least one surface of the float upon entering the vessel,contact between surfaces of the float and the second liquid allowing thesecond liquid to form a layer above the first liquid thereby formingseparate layers of liquid; and

repeating the second introducing step with successive introducing stepswith a third, fourth and so on liquid.

The float used in the above method slows the velocity of fluid such thatflow of liquid is laminar thereby limiting mixing of the two liquids.

In accordance with another aspect of the present invention, an apparatusfor producing liquid density gradients includes a vessel and a floatpositionable in the vessel. The float is formed with at least onesurface that is shaped to inhibit acceleration of fluid introduced intothe vessel thereby restricting turbulent flow of the fluid.

An outer peripheral surface of the float and the inner surface of thevessel are sized such that in response to fluid being introduced intothe vessel above the float, the fluid undergoes capillary action movingdownward beneath the float in the vessel.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages of the disclosed invention will become apparent froma reading of the following description when read in conjunction with theaccompanying drawings where like reference numerals are used to identifylike parts, in which:

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G are side views of a vessel and afloat for producing a density gradient in the vessel in accordance withthe present invention;

FIGS. 2A, 2B and 2C show details of the design and operation of float;

FIGS. 3A, 3B and 3C are side views showing alternate embodiments of thefloat;

FIGS. 4A and 4B are a side view showing yet another embodiment of thefloat; and

FIG. 5 is a side view showing still another embodiment of the float.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the present invention is illustrated in FIGS. 1A,1B, 1C, 1D, 1E, 1F, and 1G. In accordance with the present invention, afloat 1 is used to form a step gradient within a vessel 2, as depictedin FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. However, it should beunderstood that the float 1 may also be used to create a continuousgradient (not shown) where a density gradient is introduced gradually,and continuously changes along the height of the vessel. In FIG. 1A thefloat 1 in the vessel 2 rises and floats on top of a liquid 3 introducedfrom a source 4. The diameter of the float 1 in the first embodiment ispreferably slightly smaller than the inside diameter of the vessel 2.

The introduced liquid 3 contacts and then flows around the float 1,passes down between the outer surface of the float 1 and the innersurface of the vessel 2 and, as shown in FIG. 1A, produces a first zone5. Typically, the zone 5 has the highest density of all the layers orzones, as is described in greater detail below. As shown in FIG. 1B, asecond liquid 6 is introduced in a similar manner to produce zone 7. Asshown in FIGS. 1C, 1D, and 1E, the procedure is repeated with succeedingless dense liquids 8, 10, and 12, to produce zones 9, 11, and 13.

A sample 14 to be analyzed within the vessel 2 is introduced last from apipette 15 to produce zone 16 as shown in FIGS. 1F and 1G. Finally asshown in FIG. 1G, the float 1 is removed by grasping a projecting pin 17and lifting. The vessel 2 depicted in FIG. 1G with sample and gradientmay then be subjected to treatment by, for instance, insertion into aswinging bucket rotor of a centrifuge device. When a continuous gradientis required, the step gradient is prepared beforehand, and diffusion fora determined period of time used to convert the step gradient into acontinuous one.

FIGS. 2A, 2B and 2C illustrate details of the float 1 and basicprinciples of operation of the float within the vessel 2. The float 1and vessel 2 depicted in FIG. 2A is shown enlarged in FIG. 2B. The float1 has an outer peripheral surface 20 that is shaped to conform to aninner surface of the vessel 2. For instance, the vessel 2 shown in thedrawings is a tube having a circular cross-section when viewed from topor bottom. The float 1 has a corresponding circular shape with the outerperipheral surface 20 having a diameter that is smaller than the innerdiameter of the surface of the vessel 2. Therefore, a gap G having apredetermined width is defined between the outer peripheral surface 20of the float 1 and the inner surface of the vessel 2. The gap G may varyin size depending upon the solutions to be introduced into the vessel 2and the relative sizes of the float 1 and vessel 2.

In the depicted embodiment, the gap G is relatively small such thatsurface tension of the solution produces a capillary action within thegap G to maintain liquid in the gap and to prevent air bubbles in thegap. In many applications of the present invention the viscosity offluid in the gap is sufficient to eliminate the possibility of turbulentfluid flow within the vessel 2 as the solution exits the gap and movesaround the float 1. Therefore, mixing of layers of solution under thegap is almost non-existent and very sharp boundaries are producedbetween the zones, even with very small density increments.

It should be understood that diminished rate of fluid flow is adesirable result of the present invention depicted in FIGS. 1A-1G andFIGS. 2A-2C. The actual size of the gap G may be varied according to theviscosity of the liquids used and the size of the vessel 2. However,although capillary action and restricted rate of fluid flow areimportant to the present invention, it is possible to use the float 1 ofthe present invention without capillary action. For instance, the shapeof the surfaces of the float 1 may be formed to discourage any increasesin velocity of fluid moving over the surfaces of the float 1 to avoidturbulent flow of the fluids entering the vessel 2. The shape andsurface contours of the float 1 are such that the flow of solutionaround the float 1 as the solution moves downward into the vessel 2 isminimal. Specifically, a upper surface 22 of the float 1 is taperedhaving a conical shape such that as fluid contacts the upper surface 22viscous flow slows fluid motion as the fluid approaches an edge 23 ofthe float 1.

It should be understood that the upper surface 22 may have a morerounded shape when viewed from the side and need not be conical in shapeso long as sufficient surface area is provided to allow the adhesiveforces of the fluid to make contact with the upper surface 22 to slowmovement of the fluid.

It should also be understood that the vessel 2 and float 1 may have anyof a variety of shapes when viewed in cross-section. The depicted vessel2 is a tube having a circular cross-section. The vessel 2 may also havea square or triangular cross-sectional shape and the float 1 acorresponding square or triangular cross-sectional shape.

As shown in FIG. 2B, when a droplet 21 of solution is dropped from abovethe float 1, the droplet 21 is distributed circumferentially on uppertapered surface 22 and moves toward the edge 23, where the solutionflows evenly into the gap G, and thereafter slowly moves on to the uppersurface of the underlying layer of liquid. Velocity or speed of flow ofthe solution is also further decelerated as it flows around lower taper25. Capillary forces and solution viscosity are sufficient to keep thevelocity of the solution in the gap G to a minimum and further,regardless of the density of the liquids used, the gap G typicallyremains filled with solution due to the capillary action.

The float 1 is also formed with an upper integral pin 17 that allows thefloat 1 to be inserted and removed easily from the vessel 2. The densityof the float 1 itself may be dictated by the choice of constructionmaterial or, as shown in FIG. 2C, an alternate embodiment of a float 1 amay be formed with a cavity 26 sealed by plug 27 to adjustably controlthe density of the float 1. The floats 1 and 1 a are preferentiallyconstructed of polypropylene which has a density of approximately 0.95g/cc, and the pin 17, being a small fraction of the mass of the float,may be either integrally molded into the float and of the same material,or may be another material such as polycarbonate or other plastic, andbe inserted in a hole in the float as shown in FIGS. 4A and 4B. Further,the density of the float may be adjusted by inserting pins 17 having avariety of weights. For instance, a plurality of pins 17 may beproduced, each pin 17 having a different mass for selectively adjustingthe overall weight of the float.

The shape of the various surfaces of the float 1 is not limited to thedepictions in FIGS. 1A-1G and FIGS. 2A-2C. FIGS. 3A, 3B and 3Cillustrate alternative float designs. In FIG. 3A a float 28 has upperedges 29 and lower edges 30 rounded to further assist in slowlyaccelerating flow at the upper edge, and decelerating flow at the loweredge as liquid flows over the underlying liquid. In FIG. 3B float 31 hasdifferent upper edge 32 and lower edge 33, with the upper edge 32 sharpto help prevent air bubbles between the float and tube 2, and the loweredge 33 well rounded, while in FIG. 3C float 34 has a tip 35 extended tofurther control flow around the float. The shape of the lower surface ofthe float 34 and the tip 35 assist in keeping the interface 36 betweentwo steps in the gradient sharp.

The inventors have tested and designed floats for Beckman Ultracleartubes for the Beckman SW41 Ti rotor and for polycarbonate tubes for theBeckman SW28 rotor. For the SW 41 tubes, the floats were constructed ofsolid polypropylene, 13.1 mm in diameter with top and bottom tapers of15 degrees, and were 6.35 mm high measured at the edge. Wall clearancewas 0.25 mm (gap G). For the SW 28 rotor tubes, long and short versionsof the floats were constructed which were 10.5 mm and 6 mm high at theedge, had clearances of 1 and 0.6 mm, with 15 degree tapers at the topand bottom. Holes through the float had 1.6 mm internal diameters, andthe pins were made of 0.9 mm outside diameter polycarbonatemonofilament. After the pins were inserted, one end was melted in areducing flame to produce a ball at the tip, while the other end washeated to produce a small enlargement, which, when put into the float,sealed the pin in place.

All radial clearances kept the gap G between the float and thecentrifuge tube wall (vessel 2) full of liquid at all densities used.Occasionally when floats were dropped into dry tubes, they became stuckat the bottom, hence the “round” at the bottom of the centrifuge tube ispreferably filled with a “cushion”, i.e., densest gradient solutionused, initially.

Experimentally it was found that if the first 4-5 drops (circa 0.1 ml)of the solution being added were introduced slowly over a period of 5-10seconds, extraordinarily sharp interfaces were produced below the float.The remainder of the gradient step could then be introduced morerapidly. Sharp interfaces were produced with the density differencebetween two steps being as little as 0.0017 g/ml.

The use of floats allows gradients to be formed as a series of shortwell defined zones that may be arranged to be linear, sigmoidal, or ofother gradient shape. If required, the gradients can then be evened bydiffusion. The float/vessel arrangement allows the production ofgradients that are more reproducible than those produced by conventionalgradient makers, and allows many gradients to be made in parallelwithout requiring a multiplicity of gradient makers.

However, it should also be understood that by using the float of thepresent invention, it is possible to quickly pour an amount of a fluiddirectly onto the top of the float and the fluid will gradually seepdown around the float to create a layer fluid without significantlydisturbing the layer or layers of fluid already beneath the float.Without the float, pouring of fluid into the vessel with previouslyintroduced fluid layers in the vessel would guarantee mixing of thelayers thereby making gradient layer formation impossible. Therefore,one important result possible by using any of the above describedembodiments of the present invention is that a density gradient can beproduced quickly and reproducibly without concern of the rate of flow ofany one liquid onto the upper surface of the float.

In yet another embodiment of the present invention depicted in FIG. 5, afloat 50 is positioned in a vessel 52 with the vessel 52 having an innerdiameter that is significantly greater than the outer diameter of thefloat 50. The float 50 is similar to the float described above in FIGS.1A-1G, but has a tube 55 attached to an upper surface of the float 50.The tube 55 is hollow and includes several apertures 58 for allowing theflow of fluid from within the tube 55 to an upper surface of the float50. As fluid is introduced from the tube 55 via the apertures 58, thefluid contacts the upper surface of the float 50 and flows along theupper surface due to adhesion thereby slowly entering the vessel.Adhesion between the surfaces of the float 50 and the fluid slowsvelocity of the fluid such that the fluid forms a well defined layerabove previously introduced layers.

The upper surface 51 of the float 50 is preferably formed with only aslight incline to further inhibit acceleration of the fluid. The tube 55attached to the fluid may also be used to raise and lower the float 50with respect to the vessel 52. Specifically, a mechanical arm may beattached to the tube 55 to remotely control movement of the float in andout of the vessel 51. It should be understood that the tube 55 isflexible to allow movement of the float 50 upward as the vessel 52 isfilled with fluid.

A fluid flow controller (not shown) is preferably used with theembodiment of the float 50 to control the amount of fluid introduced foreach desired layer.

It should be understood that gradients may be used in many applicationsoutside of the field of molecular biology. For instance, a vessel havinga plurality of layers solution, each layer having a different densitydue to a specific concentration of solute in each layer, may be useddetermine the density of an unknown material. A sample of material ofunknown density dropped into the vessel will settle in the layer havinga like density thereby providing a means for determining density of theunknown material. For example, different classes of plastics havedifferent densities. Small pieces of plastic may easily be tested bydropping one small sample into a vessel having a plurality of solutions,each solution having a predetermined density such that the plurality oflayers define a stepped density gradient. The plastic particle will dropto a layer having the same density and will float above those layershaving a heavier density. Similarly, identification of a gemstone basedon density can be conducted.

The materials and methods of the instant invention can be used in theseparation of cellular elements from samples of whole blood, bloodproducts or diluted blood. For example, white blood cells can beobtained from blood by density gradient centrifugation. Suitablematerials to effect separation of the cellular elements and particularlythe nucleated cellular elements from blood include media that comprisecolloidal silica, silica gel, sugars, such as sucrose, ficoll, andparticular products such as Ficoll-Hypaque, Isopaque, LymphoPrep andPercoll. See, for example, Parish et al., Eur. J. Imm. (1974) 4:808.

Generally single step gradients are produced by gently layering theblood cell suspension onto a high density medium. The preparation thenis centrifuged at low speed to effect separation of the cells.

Alternatively, the blood cell suspension can be layered onto a lineargradient, for example; of bovine serum albumin prior to centrifugation.The blood cell suspension can be layered onto a discontinuous gradient,for example, of bovine serum albumin. The densities of the layers can beconfigured so that the various elements band at the interfaces of thelayers.

To ensure that discrete, sharp layers and hence tight banding of cellsoccurs, it is beneficial to ensure a sharp interface between the cellsuspension, for example, blood, and the separation medium. That goal canbe achieved with use of an apparatus of interest. A suitably sized floatof interest is used. The float of interest rests atop the separationmedium. The float of interest allows passage of the, for example, bloodalong the lateral sides thereof and along the inner surface of thecentrifuge tube containing the medium, float and cell suspension withminimal turbulence to ensure formation of a discrete linear interface ofcell suspension and separation medium.

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All references cited herein are herein incorporated by reference inentirety.

Although the present invention has been described with reference to thepreferred embodiments, the invention is not limited to the detailsthereof. Various substitutions and modifications will occur to those ofordinary skill in the art and all such substitutions and modificationsare intended to fall within the scope of the invention as defined in theappended claims.

What is claimed is:
 1. A method for isolating nucleated cells from bloodcomprising; (a) introducing a blood sample into a vessel containingblood cell separating medium and a float being formed with at least onesurface that is shaped to inhibit acceleration of a blood sampleintroduced into said vessel, thereby restricting turbulent flow of saidblood sample onto said separating medium; (b) allowing said float torise to the top of said sample and for said blood sample to contact saidblood cell separating medium; and (c)centrifuging said sample in saidvessel to produce a gradient, wherein nucleated cells separate and forma discrete layer in said gradient and wherein the orientation andposition of said at least one surface in said vessel is effected byparallel sides perpendicular to said surface.
 2. A method of separatingnucleated cells from a sample comprising blood, comprising: (a)introducing a blood separation medium into a vessel with parallel walls;(b) inserting a float into said vessel, wherein said float has a shapethat conforms to an inner surface of said vessel with a spacetherebetween; (c) introducing said sample into said vessel onto an upperexposed surface of said float and allowing the sample to pass by saidfloat as said float floats, wherein said space enables said sample topass by said float to form without turbulence a layer having aninterface with said medium; (d) removing said float or allowing saidfloat to stay on top of said sample until after centrifugation and thenremoving said float; (e) centrifuging said vessel; and (f) removingnucleated cells of blood contained in a layer at or about saidinterface.
 3. The method of claim 2, wherein an outer peripheral surfaceof said float and an inner opposing surface of said vessel have a roundshape.
 4. The method of claim 2, wherein step (b) is preformed prior tostep (a).
 5. The method of claim 2, wherein said medium comprisessilica.
 6. The method of claim 2, wherein said medium comprises acopolymer of sucrose and epichlorohydrin.
 7. The method of claim 2,wherein said sample is diluted prior to step (c).